Method of treating AML subtypes using arginine-depleting agents

ABSTRACT

The invention provides a method for treating acute myeloid leukemia (AML) in a subject in need thereof, said method comprising administering a therapeutically effective amount of an arginine-depleting agent to the subject, wherein the AML is of the French-American-British (FAB) subtype M0 (undifferentiated acute myeloblastic leukemia), M2 (acute myeloblastic leukemia with maturation), M4 (acute myeloblastic leukemia with maturation), M4 eos (acute myelomonocytic leukemia with eosinophilia), M5 (acute monocytic leukemia), M6 (acute erythroid leukemia) or M7 (acute megakaryoblastic leukemia).

TECHNICAL FIELD

The present invention relates generally to the fields of biology andmedicine, and more specifically to methods for treating acute myeloidleukemia (AML). Still more specifically, the present invention relatesto methods for treating AML subtypes using arginine-depleting agents andthe use of arginine-depleting agents in the manufacture of medicamentsfor the treatment of AML.

BACKGROUND

The following discussion of the background of the invention is merelyprovided to aid the reader in understanding the invention, and is notadmitted to describe or constitute prior art to the invention.

Acute myeloid leukemia (AML), also known as acute myelogenous leukemia,is a genetically heterogeneous aggressive cancer in which theaccumulation of genetic alterations results in uncontrolled clonalproliferation of myeloid progenitor cells in the bone marrow and blood.More severe cases involve the infiltration of organs by the abnormalcells. AML is one of the most common acute leukemias in adults andchildren, accounting for approximately 80% of adult cases andapproximately 20% of childhood leukemia diagnoses. Most cases of AMLoccur in adults, with the average age of diagnosis being 68 years. Thefive-year survival rate for people over the age of 20 diagnosed with AMLis only 25%.

AML is a heterogeneous disease which is classified into severalsubtypes. There are two major classification systems for AMLsubtypes—the French-American-British (FAB) system and the World HealthOrganization (WHO) classification system. The FAB classification systemis the one most commonly used and is the one referred to herein. Mostpeople diagnosed with AML have one of nine different FAB subtypes of AML(M0, M1, M2, M3, M4, M4 eos, M5, M6 & M7). The prognosis of a case ofAML is often dependent, inter alia, on the FAB AML subtype.

Despite technological advances and an emerging understanding of thedisease, overall survival rates of patients diagnosed with AML haveplateaued and people continue to die of the disease in significantnumbers. Chemotherapy is currently the main mode of treatment for AMLand includes two main phases: induction and consolidation. Inductiontherapy aims for complete remission of the cancer. Consolidation is aterm given to post-remission therapy. Patients may die within a few daysof the commencement of treatment due to treatment-related mortality. Themajor reason patients are not cured is resistance to treatment, whichoften manifests as a relapse from remission. However, there is nocurrent standard of care for adult relapsed or refractory AML, and theprognosis in such patients is generally poor.

AML remains a challenging illness and a need exists for new therapeuticapproaches for the treatment of this aggressive cancer.

SUMMARY

The present invention provides methods for treating acute myeloidleukemia (AML) using arginine-depleting agents and the use ofarginine-depleting agents in the manufacture of medicaments for thetreatment of AML.

The inventors of the present invention have surprisingly found thatcertain FAB AML subtypes respond markedly better to arginine depletionthan others. The methods and uses described herein may therefore beuseful for targeted treatment of AML based on FAB AML subtype.

In a first aspect, the present invention provides a method for treatingacute myeloid leukemia (AML) in a subject in need thereof, said methodcomprising administering a therapeutically effective amount of anarginine-depleting agent to the subject, wherein the AML is of theFrench-American-British (FAB) subtype M0 (undifferentiated acutemyeloblastic leukemia), M2 (acute myeloblastic leukemia withmaturation), M4 (acute myeloblastic leukemia with maturation), M4 eos(acute myelomonocytic leukemia with eosinophilia), M5 (acute monocyticleukemia), M6 (acute erythroid leukemia) or M7 (acute megakaryoblasticleukemia).

In one embodiment of the first aspect, the arginine-depleting agentcomprises an arginine catabolic enzyme.

In one embodiment of the first aspect, the arginine catabolic enzyme isan arginine deiminase, arginase, arginine decarboxylase or arginine2-monooxygenase.

In one embodiment of the first aspect, the arginine-depleting agent is asynthetic arginine-depleting agent.

In one embodiment of the first aspect, the arginine-depleting agentcomprises human serum albumin, an albumin binding domain, an Fe regionof an immunoglobulin, a polyethylene glycol (PEG) group, humantransferrin, XTEN, a proline-alanine-serine polymer (PAS), anelastin-like polypeptide (ELP), a homo-amino-acid polymer (HAP),artificial gelatin-like protein (GLK), a carboxy-terminal peptide (CTP),or a combination thereof.

In one embodiment of the first aspect, the arginine-depleting agentcomprises human serum albumin, an albumin binding domain, or acombination thereof.

In one embodiment of the first aspect, the arginine-depleting agentcomprises or consists of an amino acid sequence having at least 95%,96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1.

In one embodiment of the first aspect, the arginine-depleting agentcomprises or consists of an amino acid sequence as defined in SEQ ID NO:1.

In one embodiment of the first aspect, the AML is of the FAB subtype M4or M7.

In one embodiment of the first aspect, the AML is of the FAB subtype M7and the arginine-depleting agent comprises or consists of an amino acidsequence having at least 95%, 96%, 97%, 98% or 99% sequence identity toSEQ ID NO: 1.

In one embodiment of the first aspect, the AML is of the FAB subtype M7and the arginine-depleting agent comprises or consists of an amino acidsequence as defined in SEQ ID NO: 1.

In one embodiment of the first aspect, the AML is auxotrophic forarginine.

In one embodiment of the first aspect, the arginine-depleting agent isadministered intramuscularly, intravenously, subcutaneously or orally.

In one embodiment of the first aspect, the arginine-depleting agent isadministered intravenously.

In one embodiment of the first aspect, the subject is human.

Definitions

Certain terms are used herein which shall have the meanings set forth asfollows.

As used in this application, the singular form “a”, “an” and “the”include plural references unless the context clearly dictates otherwise.

As used herein, the term “comprising” means “including”, in anon-exhaustive sense. Variations of the word “comprising”, such as“comprise” and “comprises” have correspondingly varied meanings.

As used herein, the term “plurality” means more than one. In certainspecific aspects or embodiments, a plurality may mean 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, or more, and any numerical valuederivable therein, and any range derivable therein.

As used herein, the term “between” when used in reference to a range ofnumerical values encompasses the numerical values at each endpoint ofthe range.

As used herein, the term “synthetic”, when used to describe a product,refers to a product produced by human agency as opposed to a naturallyoccurring product. For example, a “synthetic” arginine-depleting agentrefers to an arginine-depleting agent which has been produced byartificial chemical reactions.

As used herein, the terms “treat”, “treating”, “treatment”, and the likerefer to reducing or ameliorating a disorder/disease and/or symptomsassociated therewith. It will be appreciated, although not precluded,that treating a disorder or condition does not require that thedisorder, condition, or symptoms associated therewith be completelyeliminated.

As used herein, the term “catabolism” or “catabolic” refers to achemical reaction in which a molecule decomposes into other, e.g.,smaller, molecules. For example, the term “arginine catabolic enzyme”includes any enzyme capable of reacting with arginine therebytransforming it into other molecules, such as ornithine, citrulline, andagmatine.

As used herein, the term “subject” refers to any animal (e.g., amammal), including, but not limited to, humans, non-human primates,canines, felines, and rodents.

As used herein, the terms “FAB subtype”, “FAB AML subtype” and “FAB AML”refer to a subtype of AML as classified by the French-American-British(FAB) classification system. The FAB system divides AML into ninesubtypes, M0, M1, M2, M3, M4, M4 eos, M5, M6 and M7, based on the typeof cell the leukemia develops from and how mature the cells are.

As used herein, a percentage of “sequence identity” will be understoodto arise from a comparison of two sequences in which they are aligned togive a maximum correlation between the sequences. This may includeinserting “gaps” in either one or both sequences to enhance the degreeof alignment. The percentage of sequence identity may then be determinedover the length of each of the sequences being compared. For example, anucleotide sequence (“subject sequence”) having at least 95% “sequenceidentity” with another nucleotide sequence (“query sequence”) isintended to mean that the subject sequence is identical to the querysequence except that the subject sequence may include up to fivenucleotide alterations per 100 nucleotides of the query sequence. Inother words, to obtain a nucleotide sequence of at least 95% sequenceidentity to a query sequence, up to 5% (i.e. 5 in 100) of thenucleotides in the subject sequence may be inserted or substituted withanother nucleotide or deleted.

As used herein, the term “auxotrophic”, when used to describe a cancer,refers to a cancer which is unable to synthesize one or more specificsubstances required for growth and/or metabolism. For example, an AMLwhich is “auxotrophic for arginine” refers to an AML which is unable tosynthesize arginine.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other aspects and embodiments of the present disclosurewill become apparent from the following description of the disclosure,when taken in conjunction with the accompanying drawings, in which:

FIG. 1 provides a schematic of the relationship betweenargininosuccinate synthetase (ASS) and the urea cycle.

FIG. 2 is an image of a gel showing expression levels of autophagic(LC3-II, BECLIN-1 and phospho-AMPKα) and apoptotic (PARP-1) markers in apancreatic cell line (Mia PaCa-2) after NEI-01 treatment.

FIG. 3 is an image of a gel showing expression levels of autophagic(LC3-II, p62, phospho-AMPK-α, AMPK-α) and apoptotic (Caspase-9) markersin an AML cell line (HL-60) after NEI-01 treatment.

FIG. 4 provides Kaplan-Meier survival curves of mice with AML in a C1498(M4) syngeneic AML model.

FIG. 5A provides images of the tumour burden monitored and quantifiedusing in vivo bioluminescence imaging in a C1498 (M4) syngeneic AMLmodel.

FIG. 5B provides images of the tumour burden monitored and quantifiedusing in vivo bioluminescence imaging in a C1498 (M4) syngeneic AMLmodel.

FIG. 5C provides images of the tumour burden monitored and quantifiedusing in vivo bioluminescence imaging in a C1498 (M4) syngeneic AMLmodel.

FIG. 5D provides images of the tumour burden monitored and quantifiedusing in vivo bioluminescence imaging in a C1498 (M4) syngeneic AMLmodel.

FIG. 6 provides a graph of the tumour burden monitored and quantifiedusing in vivo bioluminescence imaging in a C1498 (M4) syngeneic AMLmodel.

FIG. 7 provides graphs showing the inhibition of tumour growth followingrepeated NEI-01 treatments in a KG-1-Derived Acute Myeloid Leukaemia(FAB AML M0) Xenograft Model. (A) shows the change in average tumourvolume in 4 weeks. (B) shows the change in average T/C % over 4 weeks.Tumour volume was measured every 3 days. By day 28, a 39% reduction wasobserved in the treatment group. Statistics were calculated using RMtwo-way ANOVA, followed by Sidak's multiple comparison for post-hocanalysis. ** indicates a p-value of less than 0.01. *** indicates ap-value of less than 0.001.

FIG. 8 provides a graph showing bioluminescence signals in micetransplanted with HL-60-gfphi-Luc+ AML cells. In vivo BLI was performedtwice a week and the changes in BLI intensity were plotted. Data areexpressed as mean±SEM.

FIG. 9 provides graphs which show the inhibition of tumour growthfollowing repeated NEI-01 treatments in a P31/FUJ-Derived Acute MyeloidLeukaemia (FAB AML M5) Xenograft Model. (A) shows the change in averagetumour volume in 4 weeks. (B) shows the change in average T/C % over 4weeks. Statistics were calculated using RM two-way ANOVA, followed bySidak's multiple comparison for post-hoc analysis. * indicates a p-valueof less than 0.05. ** indicates a p-value of less than 0.01. ***indicates a p-value of less than 0.001. (C) shows the difference intumour weight between control and treated groups.

FIG. 10 provides graphs which show the inhibition of tumour growthfollowing repeated NEI-01 treatments in a MKPL-1-Derived Acute MyeloidLeukaemia (FAB AML M7) Xenograft Model. (A) shows the change in averagetumor volume in 3 weeks. (B) provides the change in average T/C % over 3weeks. (C) shows the tumor weight after the termination of the study.Statistics were calculated using RM two-way ANOVA, followed by Sidak'smultiple comparison for post-hoc analysis. * indicates a p-value of lessthan 0.05. *** indicates a p-value of less than 0.001.

FIG. 11 provides a graph of growth curves of tumour burden (presented by% of hCD45+ cells) in peripheral blood. Data is expressed as mean±SEM.

FIG. 12 is a graph of Kaplan-Meirer survival curves of mice in an AM8096model.

FIG. 13 provides graphs showing the in vivo response of Jurkat cells toNEI-01. (A) shows the change in tumour volume (%) in 4 weeks. (B) showsthe change in average T/C % in 4 weeks. Tumour volume was measured twicea week. Data are expressed as mean±SEM. A two-tailed student T-test wasused. * indicates a p value of equal to or less than 0.05.

FIG. 14A provides graphs of the mean plasma concentration of NEI-01 inmale mice and for a Repeated Dose Study on Day 1 and Day 22.

FIG. 14B provides graphs of the mean plasma concentration of NEI-01 infemale mice for a Repeated Dose Study on Day 1 and Day 22.

DETAILED DESCRIPTION

Acute myeloid leukemia (AML) is one of the most common acute leukemiasin adults and children. Current methods for treating AML are sometimesresponsible for treatment-related mortality. In cases where treatmentachieves initial success, relapse from remission is common. It is highlyimportant that new therapeutic approaches are developed for thisaggressive cancer.

The present invention provides methods for treating AML usingarginine-depleting agents. The methods provided herein may reduce orameliorate the disease and/or symptoms associated therewith. The methodsmay or may not completely eliminate said disease and/or symptoms. Thearginine-depleting agents described herein may also be used for themanufacture of medicaments for the treatment of AML, which may reduce orameliorate the disease and/or symptoms associated therewith, and may ormay not completely eliminate said disease and/or symptoms.

FAB AML Subtypes

AML is a heterogeneous disease which is classified into severalsubtypes. There are two major classification systems for AMLsubtypes—the French-American-British (FAB) system and the World HealthOrganization (WHO) classification system. The FAB classification systemis the one most commonly used and is the one referred to herein. Mostpeople diagnosed with AML have one of nine different FAB subtypes of AML(M0, M1, M2, M3, M4, M4 eos, M5, M6 & M7). The prognosis of a case ofAML is often dependent, inter alia, on the AML subtype.

The FAB classification system divides AML into subtypes M0 to M7 basedon the type of cell the leukemia develops from and how mature the cellsare, as outlined in Table 1:

TABLE 1 FAB AML subtypes and descriptions FAB subtype Description M0Undifferentiated acute myeloblastic leukemia M1 Acute myeloblasticleukemia with minimal maturation M2 Acute myeloblastic leukemia withmaturation M3 Acute promyelocytic leukemia M4 Acute myelomonocyticleukemia M4 eos Acute myelomonocytic leukemia with eosinophilia M5 Acutemonocytic leukemia M6 Acute erythroid leukemia M7 Acute megakaryoblasticleukemia

The present invention provides methods for treating AML and the use ofarginine-depleting agents for the manufacture of a medicament for thetreatment of AML of any subtype. In some embodiments, the inventionprovides methods, and the use of arginine-depleting agents for themanufacture of medicaments for treating FAB AML M0. The methods andmedicaments of the present invention may also be used to treat FAB AMLM2. In other embodiments, the invention provides methods and medicamentsfor treating FAB AML M4. In further embodiments, the invention providesmethods and medicaments for treating FAB AML M4 eos. In still furtherembodiments, the invention provides methods and medicaments for treatingFAB AML M5. Methods and medicaments for treating FAB AML M6 are alsoprovided herein. The present invention also provides methods andmedicaments for treating FAB AML M7. Although the methods andmedicaments provided herein are presented in the context of treating AMLsubtypes as defined by the FAB classification system, the skilled personwill understand that they may be used to treat cases of AML classifiedusing any other system or method of classification.

The FAB AML classification system was established in 1976 and is wellknown in the art. A person skilled in the art can easily identify theFAB AML subtype of a sample using, for example, histochemical stainingand microscopy. The AML sample used may be obtained, for example, fromperipheral blood, a bone marrow aspirate or a biopsy. A detaileddescription of each FAB AML subtype, including images to aididentification, is provided in Charles A. Schiffer, MD and Richard M.Stone, MD (2003) in “Holland-Frei Cancer Medicine, 6th edition”, Kufe DW, Pollock R E, Weichselbaum R R, et al. (eds.) Hamilton (ON), 1983.

Arginine is required for a variety of metabolic pathways. Many tumoursare auxotrophic for arginine due to low levels or the absence ofargininosuccinate synthetase (ASS) and/or ornithine transcarbamoylase(OTC), which are required for arginine synthesis. In most cases of AML,the cells are deficient in ASS1, the gene encoding ASS in humans. Itwould be easy for a person skilled in the art to determine whether thecells from an AML sample were deficient in one or both of theaforementioned enzymes using well-known methods such as Western Blot,ELISA SDS-PAGE or immunoprecipitation.

Some embodiments of the present invention provide methods for treatingAML in a subject comprising administering a therapeutically effectiveamount of an arginine-depleting agent to the subject. Furtherembodiments provide the use of arginine-depleting agents for themanufacture of a medicament for the treatment of AML in a subject inneed thereof. The subject may be any animal (e.g., a mammal), including,but not limited to, humans, non-human primates, canines, felines, androdents.

Arginine-Depleting Agents

An arginine-depleting agent used in the treatment methods andmedicaments described herein may be any arginine-depleting agent knownin the art to be capable of reducing plasma and/or cellular levels ofarginine in a subject. The arginine-depleting agent may, for example, bea small molecule or protein.

In some embodiments, the arginine-depleting agent comprises an argininecatabolic enzyme. Non-limiting examples of arginine catabolic enzymesinclude arginase, arginine deiminase, arginine decarboxylase andarginine 2-monooxygenase.

The arginase may be any arginase known in the art, such as thoseproduced by bacteria, fungi, fish, humans, bovines, swine, rabbits,rodents, primates, sheep and goats. Non-limiting examples of arginasesinclude Bacillus caldovelox arginase, Thermus thermophilus arginase,Capra hircus arginase I, Heterocephalus glaber arginase I, Bos taurusarginase I, Sus scrofa arginase I, Plecoglossus altivelis arginase I,Salmo salar arginase I, Oncorhynchus mykiss arginase I, Osmerus mordaxarginase I, Hyriopsis cumingii arginase I, Rattus norvegicus arginase I,Mus musculus arginase I, Homo sapiens (human) arginase I, Pantroglodytes arginase I, Oryctolagus cuniculus arginase I, Rattusnorvegicus arginase II, Mus musculus arginase II, Homo sapiens (human)arginase II, Bostaurus arginase II, Heterocephalus glaber arginase II,Pan troglodytes arginase II, Oryctolagus cuniculus arginase II, Delftiaarginase, Bacillus coagulans arginase, Hoeflea phototrophica arginaseand Roseiflexus castenholzii arginase. Other examples include arginasesfrom Bacillus methanolicus, Bacillus sp. NRRL B-14911, Planococcusdonghaensis, Paenibacillus dendritiformis, Desmospora sp., Methylobactertundripaludum, Stenotrophomonas sp., Microbacterium laevaniformans,Porphyromonas uenonis, Agrobacterium sp., Octadecabacter arcticus,Agrobacterium tumefaciens, Anoxybacillus flavithermus, Bacillus pumilus,Geobacillus thermoglucosidasius, Geobacillus thermoglucosidans,Brevibacillus laterosporus, Desulfotomaculum ruminis, Geobacilluskaustophilus, Geobacillus thermoleovorans, Geobacillusthermodenitrificans, Staphylococcus aureus, Halophilic archaeon DL31,Halopigerxanaduensis, Natrialba magadii, Plasmodium falciparum,Helicobacter pylori, and the like.

An arginine deiminase used in the methods and medicaments of the presentinvention may be any arginine deiminase known in the art, such as thoseproduced from Mycoplasma, Lactococcus, Pseudomonas, Steptococcus,Escherichia, Mycobacterium or Bacillus microorganisms. Exemplaryarginine deiminases include, but are not limited, to those produced byMycoplasma hominis, Mycoplasma arginini, Mycoplasma arthritidis,Clostridium perfringens, Bacillus licheniformis, Borrelia burgdorferi,Borrelia afzellii, Enterococcus faecalis, Lactococcus lactis, Bacilluscereus, Streptococcus pyogenes, Steptococcus pneumoniae, Lactobacillussake, Giardia intestinalis, Mycobacterium tuberculosis, Pseudomonasplecoglossicida, Pseudomonas putida, Pseudomonas aeruginosa, and thelike.

The arginine decarboxylase may be any arginine decarboxylase known inthe art, such as those produced by Escherichia coli., Salmonellatyphimurium, Chlamydophila pneumoniae, Methanocaldococcus jannaschii,Paramecium bursaria Chlorella virus 1, Vibrio vulnificus YJ016,Campylobacter jejuni subsp., Trypanosoma cruzi, Sulfolobus solfataricus,Bacillus licheniformis, Bacillus cereus, Carica papaya,Nicotianatobacum, Glycine max, Lotus coniculata, Vibrio vulnificus,Vibrio cholerae, Mus musculus, Thermotoga, Rattus norvegzcus, Homosapiens, Bos taurus, Susscrofa, Thermus thermophiles, Thermusparvatiensis, Thermus aquaticus, Thermus thermophiles, Thermusislandicus, Arabidopsis thaliana, Avena sativa, and the like.

An arginine 2-monooxygenase used in the methods and medicaments of thepresent invention may be any arginine 2-monooxygenase known in the art,such as those produced from Arthrobacter globiformis IFO 12137,Arthrobacter simplex IFO 12069, Brevibacterium helvolum IFO 12073,Helicobacter cinaedi CCUG 18818, Streptomyces griseus, and the like.

The arginine-depleting agents of the present invention may comprisenaturally occurring and/or synthetic products. In some embodiments ofthe invention, the arginine-depleting agents comprise naturallyoccurring arginine catabolic enzymes. In other embodiments, thearginine-depleting agents comprise synthetic arginine catabolic enzymes.

The arginine-depleting agents may comprise full proteins and/orfunctional fragments and/or variants thereof. Arginine decarboxylases,arginine deiminases, arginine 2-monooxygenases, arginases and otherarginine-depleting agents used in the methods and uses may be modifiedto improve their pharmacokinetic properties, such as by fusion ofproteins and/or functional fragments and/or variants thereof with humanserum albumin, an albumin binding domain, an Fe region of animmunoglobulin, a polyethylene glycol (PEG) group, or a combinationthereof. In some embodiments of the invention, one or more of theaforementioned modifications lengthens the half-life of thearginine-depleting agent. In further embodiments, the increase inhalf-life results in a reduction of the frequency of administration ofthe arginine-depleting agent required to achieve the same outcome. Oneor more of the aforementioned modifications to the arginine-depletingagents may reduce immunogenicity, which may help to avoid adverseeffects.

In some embodiments of the present invention, arginine catabolic enzymesmay be engineered to include specific sites on the enzyme where, forexample, PEG can be selectively attached. The selected PEGylation sitesmay be located at a site removed from the active site of the enzyme,and/or may be generally exposed to solvent to allow reaction withPEGylation reagents.

Any PEGylation reagent known in the art can be used to covalently attachPEG to the arginine catabolic enzymes described herein. ExemplaryPEGylation reagents include, but are not limited to mPEG-ALD(methoxypolyethylene glycol-propionaldehyde); mPEG-MAL(methoxypolyethylene glycol-maleimide); mPEG-NHS (methoxypolyethyleneglycol-N-hydroxy-succinimide); mPEG-SPA (methoxypolyethyleneglycol-succinimidyl propionate); and mPEG-CN (methoxypolyethyleneglycol-cyanuric chloride).

In some embodiments, the PEG group has a molecular weight of about 5,000to about 20,000 amu, about 5,000 to about 15,000 amu, about 5,000 toabout 12,000 amu, about 7,000 to about 12,000 amu, or about 7,000 toabout 10,000 amu. In certain embodiments, the PEG group has a molecularweight of about 2,000 amu to 10,000 amu. In some embodiments, the PEGgroup is PEG4,000, PEG5,000, PEG6,000, or PEG7,000.

The PEG group may be covalently attached directly to the enzyme orattached via a linker. In certain embodiments, the enzyme is covalentlyattached via a propionic acid linker to PEG.

Arginine catabolic enzymes may be fused to proteins with an inherentlylong serum half-life, which may result in more desirable pharmacokineticprofiles. The arginine-depleting agents of the present invention maycomprise an antibody Fc domain and/or serum albumin. Thearginine-depleting agents may comprise arginine catabolic enzymesgenetically fused to an antibody Fc domain and/or serum albumin. In someembodiments, the Fe region of an immunoglobulin is from humanimmunoglobulin, for example, human IgG. In some embodiments, the enzymesmay be fused to an albumin binding domain. In some embodiments, theenzymes may be fused to human transferrin.

In further embodiments of the invention, the arginine-depleting agentscomprise arginine catabolic enzymes fused to non-structuredpolypeptides. Fusion of the enzymes to non-structured polypeptides mayincrease the overall size and/or hydrodynamic radius of the agents. Insome embodiments of the invention, arginine catabolic enzymes are fusedto any one or more of XTEN, which is a recombinant PEG mimetic(XTENylation), PAS, which is a proline-alanine-serine polymer(PASylation), ELP, which is an elastin-like polypeptide (ELPylation),HAP, which is a homo-amino-acid polymer (HAPylation), and artificialgelatin-like protein (GLK).

The arginine catabolic enzymes used in some embodiments of the inventionmay be fused to anionic polypeptides, which may increase the negativecharge of the agents. Enzymes may be fused to a carboxy-terminal peptide(CTP). One non-limiting example of suitable CTP fusion is the geneticfusion of the CTP from the human chorionic gonadotropin (CG) β chain.

Arginine catabolic enzymes may be linked to serum albumin vianon-covalent interactions with serum albumin, which may also extend thehalf-life of the agents. In one non-limiting example of an embodiment ofthe invention, an albumin-binding moiety is either conjugated orgenetically fused to the therapeutic enzyme. Many types of moieties canbe used, including, but not limited to (i) molecules with intrinsicaffinity for albumin; (ii) peptides, antibody fragments, alternativescaffolds, and small chemicals generated and selected to exhibit albuminbinding activity.

Recombinant fusion proteins were first used in the 1980s and are createdby the fusion of two or more genes which each encode a separate protein.A variety of methods for the synthesis of fusion proteins are well knownin the art. See, for example, Yu et al., Biotechnology Advances, 2015;33: 155-164, which provides a review of the most common approachescurrently used for the design and construction of synthetic fusionproteins. Strohl, Biodrugs, 2015; 29(4): 215-239, provides anotherdetailed review of fusion proteins and outlines the advantages anddisadvantages of both fusion methods and different types of fusionprotein.

In some embodiments of the invention, the arginine-depleting agentcomprises or consists of an amino acid sequence having at least 75%,80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or100% sequence identity to SEQ ID NO: 1. In further embodiments, thearginine-depleting agent comprises or consists of an amino acid sequencehaving at least 95%, 96%, 97%, 98% 99% or 100% sequence identity to SEQID NO: 1. In still further embodiments, the arginine-depleting agentcomprises or consists of an amino acid sequence as defined in SEQ ID NO:1.

Methods for assessing the level of homology and identity betweensequences are well known in the art. The percentage of sequence identitybetween two sequences may, for example, be calculated using amathematical algorithm. A non-limiting example of a suitablemathematical algorithm is described in the publication of Karlin andcolleagues (1993, PNAS USA, 90:5873-5877). This algorithm is integratedin the BLAST (Basic Local Alignment Search Tool) family of programs (seealso Altschul et al. (1990), J. Mol. Biol. 215, 403-410 or Altschul etal. (1997), Nucleic Acids Res, 25:3389-3402) accessible via the NationalCenter for Biotechnology Information (NCBI) website homepage(https://www.ncbi.nlm.nih.gov). The BLAST program is freely accessibleat https://blast.ncbi.nlm.nih.gov/Blast.cgi. Other non-limiting examplesinclude the Clustal (http://www.clustal.org/) and FASTA (Pearson (1990),Methods Enzymol. 83, 63-98; Pearson and Lipman (1988), Proc. Natl. Acad.Sci. U. S. A 85, 2444-2448.) programs. These and other programs can beused to identify sequences which are at least to some level identical toa given input sequence. Additionally or alternatively, programsavailable in the Wisconsin Sequence Analysis Package, version 9.1(Devereux et al. 1984, Nucleic Acids Res., 387-395), for example theprograms GAP and BESTFIT, may be used to determine the percentage ofsequence identity between two polypeptide sequences. BESTFIT uses thelocal homology algorithm of Smith and Waterman (1981, J. Mol. Biol. 147,195-197) and identifies the best single region of similarity between twosequences. Where reference herein is made to an amino acid sequencesharing a specified percentage of sequence identity to a reference aminoacid sequence, the difference/s between the sequences may arisepartially or completely from conservative amino acid substitution/s. Insuch cases, the sequence identified with the conservative amino acidsubstitution/s may substantially or completely retain the samebiological activity of the reference sequence.

Administration of Arginine-Depleting Agents

For therapeutic use, the arginine-depleting agents described herein maybe prepared as pharmaceutical compositions containing a therapeuticallyeffective amount of an arginine-depleting agent described herein as anactive ingredient in a pharmaceutically acceptable carrier. The term“carrier” refers to a diluent, adjuvant, excipient, or vehicle withwhich the active compound is administered. Such vehicles can be liquids,such as water and oils, including those of petroleum, animal, vegetableor synthetic origin, such as peanut oil, soybean oil, mineral oil,sesame oil and the like. As a non-limiting example, 0.9% saline and 0.3%glycine can be used. These solutions may be sterile and generally freeof particulate matter. They may be sterilized by conventional,well-known sterilization techniques (e.g., filtration). The compositionsmay contain pharmaceutically acceptable auxiliary substances as requiredto approximate physiological conditions such as pH adjusting andbuffering agents, stabilizing, thickening, lubricating and colouringagents, etc. The concentration of the arginine-depleting agent in suchpharmaceutical formulation can vary widely and may be selected based onrequired dose, fluid volumes, viscosities, etc., according to theparticular mode of administration selected. Suitable vehicles andformulations are described, for example, in Remington: The Science andPractice of Pharmacy, 21st Edition, Troy, D. B. ed., Lipincott Williamsand Wilkins, Philadelphia, Pa. 2006, Part 5, PharmaceuticalManufacturing pp 691-1092, See especially pp. 958-989.

The concentration of plasma arginine in the subject needed to observe atherapeutic effect may vary based on numerous factors, including thecondition of the subject and/or the type and severity of the AML and/ordiet composition. The selection of the target plasma arginine levels iswell within the skill of a person of ordinary skill in the art.

The determination of the duration of treatment e.g., the duration oftime the plasma arginine concentrations are maintained in a depletedstate in the subject, is well within the skill of a person of ordinaryskill in the art. In certain embodiments, the duration of treatment ismore than 1 week, more than 2 weeks, more than 3 weeks, more than 4weeks, more than 5 weeks, more than 6 weeks, more than 7 weeks, morethan 8 weeks, more than 9 weeks, more than 10 weeks, more than 11 weeks,more than 12 weeks, more than 24 weeks, more than 28 weeks, more than 32weeks, more than 36 weeks, more than 40 weeks, more than 44 weeks, morethan 48 weeks, more than 52 weeks, or more than 56 weeks.

No limitation applies in relation to the mode of administration of thearginine-depleting agents in the methods and uses of the presentinvention. In some embodiments of the invention, the mode ofadministration of the arginine-depleting agents is intravenous. The modeof administration for therapeutic use of the arginine-depleting agentsdescribed herein may be any suitable route that delivers the agents tothe subject, such as parenteral administration, e.g., intradermal,intramuscular, intraperitoneal, intravenous and/or subcutaneous;pulmonary; transmucosal (e.g., oral, intranasal, intravaginal and/orrectal); using a formulation in a tablet, capsule, solution, suspension,powder, gel and/or particle; and contained in a syringe, an implanteddevice, osmotic pump, cartridge and/or micropump; or other meansappreciated by the skilled artisan, as well known in the art.

It will be appreciated by persons of ordinary skill in the art thatnumerous variations and/or modifications can be made to the presentinvention as disclosed in the specific embodiments without departingfrom the spirit or scope of the present invention as broadly described.The present embodiments are, therefore, to be considered in all respectsas illustrative and not restrictive.

EXAMPLES

The present invention will now be described with reference to thefollowing specific Examples, which should not be construed as in any waylimiting.

Example 1: Cytotoxicity of NEI-01 in a Range of Cancer Cell Lines

Exogenous arginine is required for growth in some argininosuccinatesynthetase (ASS)-deficient cancers. NEI-01 is a recombinantalbumin-binding arginine deiminase which can convert arginine tocitrulline & ammonia, and inhibits growth in various ASS-deficientcancers by depleting arginine (FIG. 1). Cytotoxicity assay resultsdemonstrated that NEI-01 depleted arginine and inhibited cancer cellgrowth (especially in the case of ASS1 deficient cancer cell lines) in arange of different cancer cell lines (Table 2).

TABLE 2 Cytotoxicity assay results for different cancer cell linesfollowing treatment with NEI-01. ASS1 Expression IC50 Cancer Cell LineOrigin (Protein) (μg/ml) Hepatocellular Hepa 1-6 Mouse − 0.0080Carcinoma Hep-55 1C Mouse − 0.0076 SK-HEP-1 Human − 0.0292 Colon CancerLovo Human + >10 COLO 205 Human + >10 HT-29 Human − 0.1058 Breast CancerMCF7 Human + >10 MDA-MB-231 Human − 0.0168 4T1 Mouse − 0.0389 ProstateCarcinoma 22Rv1 Human − 0.0262 Malignant A375 Human − 0.0081 MelanomaCervical Carcinoma C-33 A Human − 0.0126 Pancreatic MIA PaCa-2 Human −0.0036 Carcinoma

The results of cytotoxicity assays for different AML cell lines aftertreatment with NEI-01 are provided in Table 3.

TABLE 3 Cytotoxicity assay results for different AML cell linesfollowing treatment with NEI-01. ASS1 AML Expression IC50 Maximumsubtype Cell Line Origin (Protein) (ug/ml) inhibition M0 KG-1 Human −0.0033 83% M2 Kasumi-1 Human + 0.0350 19% M2 HL-60 Human − 0.0068 73% M5AML-193 Human − 0.0034 63% M5 THP-1 Human + 0.2316 11% M4 C1498 Mouse −0.0031 99%

Example 2: Apoptosis and Autophagy. NEI-01 Treatment of PancreaticCancer Cell Line Mia PaCa-2

To confirm the arginine deprivation-mediated reduction of cell viabilityby autophagic cell death, Mia PaCa-2 cells were treated with designatedconcentrations of NEI-01 with or without choloquine (CQ). At indicatedtime-points, cells were harvested and subjected to immunoblotting usingantibodies against several autophagic and apoptotic markers.

As shown in FIG. 2, the expression levels of autophagic markers LC3-II,BECLIN-1 and phospho-AMPKα were increased upon NEI-01 treatment,suggesting autophagy plays a role in NEI-01-induced cell death.Conversely, expressions levels of apoptotic marker PARP-1 decreased uponNEI-01 treatment, demonstrating the activation of apoptotic pathways.These results show that apoptosis and autophagy play a role in thearginine deprivation-mediated mechanism of cell death.

Example 3. NEI-01 Treatment of AML Cell Line HL-60

To further confirm the arginine deprivation-mediated reduction of cellviability by autophagic cell death, ASS1-deficient HL-60 AML cells weretreated with NEI-01 and CQ. As shown in FIG. 3, expression levels ofautophagic markers LC3-II, p62, phospho-AMPKα and AMPKα increased uponNEI-01 treatment, suggesting autophagy plays a role in NEI-01-inducedcell death. Conversely, expressions levels of apoptotic marker Caspase-9decreased following NEI-01 treatment, demonstrating the activation ofapoptotic pathways. These results show that apoptosis and autophagy playa role in the arginine deprivation-mediated mechanism of cell death.

Examples 4 to 10: Effect of NEI-01 on AML Subtypes

Two major classification systems exist for identifying AML subtypes—theFrench-American-British (FAB) system and the World Health Organization(WHO) classification system. The FAB system is the one most commonlyused and will be used herein. According to the FAB system, most peoplediagnosed with AML have one of nine different kinds (subtypes) of AML:M0, M1, M2, M3, M4, M4 eos, M5, M6 and M7.

Example 4: Anticancer Activity of NEI-01 in the C1498 Syngeneic AcuteMyeloid Leukemia (FAB AML M4) Model

This study aimed to evaluate the anticancer activity of thearginine-depriving enzyme, NEI-01 in a C1498 syngeneic AML (FAB AML M4)Model.

Murine argininosuccinate synthase (ASS1)-deficient C1498 cellsco-labeled with luciferase and green fluorescent protein (GFP) wereintravenously (i.v.) transplanted into C57BL/6 mice to establish asyngeneic AML model. The mice were randomly divided into 4 groups.Details of the 4 groups and their corresponding treatment regimens areprovided in Table 4.

TABLE 4 Groups and treatment regimens for C1498 Syngeneic Acute MyeloidLeukaemia (FAB AML M4) Model study. No. of Group Treatment Regimenanimals Duration 1 PBS, twice a week, i.v. 7 4 weeks 2 NEI-01 140 U/kg,once a week, i.v. 8 3 NEI-01 280 U/kg, once a week, i.v. 9 4 NEI-01 280U/kg, twice a week, i.v. 8

The results showed that treatment with NEI-01 significantly prolongedthe overall survival of mice with AML subtype M4 compared withPBS-treated controls (FIG. 4). The median survival day (MSD) wasprolonged from 24 days in Group 1 (control group) to 29 days in Group 2(treated with NEI-01 140 U/kg once a week, p=0.0058 vs control group).

Moreover, more than 60% of the mice in Group 3 (treated with NEI-01 280U/kg once a week) and all of the mice in Group 4 (treated with NEI-01280 U/kg twice a week) survived until the end of the experiment. Themedian survival rate was >31 days in Group 3 and Group 4 (Group 3 vscontrol, p=0.0003; Group 4 vs control, p<0.0001). Consistent with theresults observed for overall survival, treatment with NEI-01significantly reduced the total leukemia burden in addition to slowingdown disease progression (FIGS. 5 and 6). This anticancer activity ofNEI-01 was exhibited in a dose-dependence manner.

This study demonstrated a potent anticancer activity of NEI-01 in aC1498 syngeneic AML M4 model.

Example 5: Anticancer Activity of NEI-01 in a KG-1-Derived Acute MyeloidLeukaemia (FAB AML M0) Xenograft Model

In this study, the anticancer effect of NEI-01 was evaluated in a murinexenograft model.

Human argininosuccinate synthase (ASS1)-deficient M0-subtype acutemyeloid leukemia KG-1 cells were subcutaneously injected intoimmunodeficient BALB/c nude mice. When the tumour volume reached 180mm³, the mice were intravenously (i.v.) treated with buffer MHT orNEI-01 (280 U/kg) once a week. Details of the study groups and theircorresponding treatment regimens are provided in Table 5.

TABLE 5 Groups and treatment regimens for KG-1-Derived Acute MyeloidLeukaemia (FAB AML M0) Xenograft Model study. No. of Group TreatmentRegimen animals Duration 1 Control, Buffer MHT, once a week, i.v. 9 4weeks 2 NEI-01 280 U/kg, once a week, i.v. 9

The tumour volume was measured every 3 days. After 4 weeks, the mice(n=9) were sacrificed. The xenograft tumours were then dissected andindividually weighed.

The results showed that NEI-01 treatment significantly reduced thevolume of the tumours (FIG. 7). A 39% reduction was observed by Day 28.A final T/C ratio reached at 60.6%. At the termination of the study, thetumour weights were 3.41±0.53 g in the control group and 2.03±0.47 g inthe NEI-01 (280 U/kg once a week) treatment group. There was a 40.38%decrease in tumour weight.

This study demonstrated efficient anticancer activity of NEI-01 inmurine AML M0 KG-1 xenografts.

Example 6: Anticancer Activity of NEI-01 in a HL-60 Derived AcuteMyeloid Leukemia (FAB AML M2) Orthotopic Model

In this study, the anticancer activity of NEI-01 was evaluated in amurine orthotopic AML model.

Human argininosuccinate synthase (ASS1)-deficient M2-subtype acutemyeloid leukemia HL-60 cells co-labeled with luciferase and greenfluorescent protein (GFP) were intravenously (i.v.) transplanted intononobese diabetic/severe combined immunodeficiency (NOD/SCID) mice toestablish an orthotopic AML model. The mice were randomly divided into 3groups. Details of the study groups and their corresponding treatmentregimens are provided in Table 6.

TABLE 6 Groups and treatment regimens for HL-60 Derived Acute MyeloidLeukemia (FAB AML M2) Orthotopic Model study. No. of Group TreatmentRegimen animals Duration 1 PBS, twice a week, i.v. 10 4 weeks 2 NEI-01280 U/kg, once a week, i.v. 10 3 NEI-01 280 U/kg, twice a week, i.v. 10

Leukemia cells (HL-60-gfphi-Luc+ cells) were tracked and the totalleukemia burden was quantified by in vivo BLI. The disease progressionwas determined by the changes in BLI intensity. The results are shown inFIG. 8. An aggressive disease progression was found with a strong signalevident throughout the AML mice. This progression was significantlyinhibited when the mice were treated with NEI-01 either once a week(p<0.05, from Day 4 to Day 25) or twice a week (p<0.01, whole treatmentperiod).

The results demonstrated that treatment with NEI-01 efficiently depletedarginine from plasma in the mouse, resulting in inhibition of diseaseprogression as well as reduction of tumour burden in hematopoietictissues (including bone marrow and spleen). Disease progression wassignificantly inhibited when the mice were treated with NEI-01 eitheronce a week (p<0.05, from Day 4 to Day 25) or twice a week (p<0.01,whole treatment period). Particularly efficient activity was observed inthe bone marrow and spleen; the tumour burden was significantly reducedin bone marrow (p<0.0001) and spleen (p<0.005) when the mice weretreated with 280 U/kg NEI-01 twice a week.

This study demonstrated a potent anticancer activity of NEI-01 in aHL-60 orthotopic AML M2 model.

Example 7: Anticancer Activity of NEI-01 in a P31/FUJ-Derived AcuteMyeloid Leukemia (FAB AML M5) Xenograft Model

In this study, the anticancer activity of NEI-01 was evaluated in amurine xenograft model.

Human argininosuccinate synthase (ASS1)-deficient M5-subtype acutemyeloid leukemia P31/FUJ cells were subcutaneously inoculated innonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. Whenthe tumour volume reached 200 mm³, the mice were intravenously (i.v.)treated with buffer MHT or NEI-01 (280 U/kg) once a week. The tumourvolume was measured every 3-4 days. After 4 weeks, the mice (n=10) weresacrificed and the xenograft tumours were dissected and individuallyweighed. Details of the study groups and their corresponding treatmentregimens are provided in Table 7.

TABLE 7 Groups and treatment regimens for P31/FUJ-Derived Acute MyeloidLeukemia (FAB AML M5) Xenograft Model study. No. of Group TreatmentRegimen animals Duration 1 Control, Buffer MHT, once a week, i.v. 10 4weeks 2 NEI-01 280 U/kg, once a week, i.v. 10

FIG. 9 shows that NEI-01 treatment significantly reduced the tumourvolume as well as the tumour weight, resulting in a 51.27% reduction intumour volume by Day 28. A final T/C ratio reached 51.14%. At thetermination of the study, the tumour weights were 1.08±0.98 g in thecontrol group and 0.78±0.08 g in NEI-01 (280 U/kg once a week) treatmentgroup (p<0.05).

This study demonstrated efficient anticancer activity of NEI-01 inmurine AML M5 P31/FUJ xenografts.

Example 8: Anticancer Activity of NEI-01 in a MKPL-1-Derived AcuteMyeloid Leukemia (FAB AML M7) Xenograft Model

In this study, the anticancer effect of NEI-01 was evaluated in a murinexenograft model.

Human argininosuccinate synthase (ASS1)-deficient M7-subtype acutemyeloid leukemia MKPL-1 cells were subcutaneously inoculated intononobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. Whenthe tumour size reached 120 mm³, the mice were intravenously (i.v.)treated with buffer MHT or NEI-01 (280 U/kg) once a week. The tumourvolume was measured every 2-3 days. After 3 weeks, the mice (n=9) weresacrificed and the xenograft tumours were dissected and individuallyweighed. Details of the study groups and their corresponding treatmentregimens are provided in Table 8.

TABLE 8 Groups and treatment regimens for MKPL-1-Derived Acute MyeloidLeukemia (FAB AML M7) Xenograft Model study. No. of Group TreatmentRegimen animals Duration 1 Control, Buffer MHT, once a week, i.v. 9 3weeks 2 NEI-01 280 U/kg, once a week, i.v. 9

FIG. 10 shows that NEI-01 treatment significantly reduced the tumourvolume as well as the tumour weight, resulting in a 99% reduction intumour volume by Day 22. Tumour weights were 8.05±0.056 g in the controlgroup and 0.15±0.05 g in the NEI-01 (280 U/kg once a week) treatmentgroup. A final T/C ratio reached at 99%.

This study demonstrated efficient anticancer activity of NEI-01 inmurine AML M7 MKPL-1 xenografts.

Example 9: In Vivo Efficacy Study of NEI-01 in the Treatment of aPatient-Derived AM5512 Acute Myeloid Leukemia (FAB AML M7) Model

Patient-derived xenograft (PDX) offers the most translationalpreclinical model for efficacy screening in cancer drug development.Derived directly from patient tumours and never adapted to grow invitro, PDX models reflect the heterogeneity and diversity of the humanpatient population. In this study, the anticancer effect of NEI-01 wasevaluated in a Patient-Derived AM5512 (FAB AML M7) Acute MyeloidLeukemia Model.

Human AM5512 cells were intravenously (i.v.) inoculated into nonobesediabetic/severe combined immunodeficiency (NOD/SCID) mice. When thetumour burden in peripheral blood was ˜1.33%, the mice were randomlydivided into 3 groups: group 1 (Vehicle), group 2 (NEI-01, 140 U/kg) andgroup 3 (NEI-01, 280 U/kg) as outlined in Table 9.

TABLE 9 Groups and treatment regimens for Patient-Derived AM5512 AcuteMyeloid Leukemia (FAB AML M7) Model. Dose Route of Treat- level adminis-Dosing Dosing Group No. ment (U/kg) tration Frequency Duration 1 10Vehicle — i.v. Weekly on D 1, 8, 2 10 NEI-01 140 15, 22 3 10 NEI-01 280

Treatment with NEI-01 (either 280 U/kg or 140 U/kg once a week)significantly inhibited the tumour burden growth in peripheral bloodafter the 3^(rd) dose of NEI-01 (FIG. 11). At the termination of thestudy (1 week after the 4th dose of NEI-01), a significant reduction intumour burden was observed in peripheral blood and hematopoietic tissues(including spleen, liver and bone marrow) following treatment withNEI-01, either 280 U/kg or 140 U/kg once a week (p<0.05, compared tovehicle group). These anti-leukemia effects were exhibited in adose-dependent manner.

This study provides a strong evidence to support that NEI-01 has apotent anti-leukemia effect in an AM5512 (M7) PDX model.

Example 10: In Vivo Efficacy Study of NEI-01 in the Treatment of aPatient-Derived AM8096 Acute Myeloid Leukemia (FAB AML M2) Model

In this study, the anticancer effect of NEI-01 was evaluated in aPatient-Derived AM8096 Acute Myeloid Leukemia Model.

Human AM8096 cells were intravenously (i.v.) inoculated into nonobesediabetic/severe combined immunodeficiency (NOD/SCID) mice. When thetumour burden in peripheral blood was ˜1.5%, the mice were randomlydivided into 3 groups: group 1 (Vehicle), group 2 (NEI-01, 140 U/kg) andgroup 3 (NEI-01, 280 U/kg) as outlined in Table 10.

TABLE 10 Groups and treatment regimens for Patient-Derived AM8096 AcuteMyeloid Leukemia (FAB AML M2) Model study. Dose Route of Treat- leveladminis- Dosing Dosing Group No. ment (U/kg) tration Frequency Duration1 10 Vehicle — i.v. Weekly Weekly 2 10 NEI-01 140 injection 3 10 NEI-01280 until humane endpoints (on D 1, 8, 15, 22, 29, 36 . . . etc)

The results showed that treatment with NEI-01 once a week slightlyprolonged the median number of survival days from 12 days in the vehiclecontrol group to 14.5 days in the NEI-01 (140 U/kg) treatment group and16.5 days in the NEI-01 (280 U/kg) treatment group (FIG. 12 and Table11).

The increase in life-span (ILS) of NEI-01 treated mice (140 U/kg or 280U/kg) was 20.8% and 37.5% respectively when compared to vehicle controls(Table 11). Excitingly, one of the mice in the NEI-01 (280 U/kg)treatment group survived until 41 days after the initial treatment.These data suggest that NEI-01 has anti-leukemia effects at least insome populations of AM8096 models.

TABLE 11 Median survival days and life-span (ILS) for each group in theAM8096 model. Median Number of p value ILS survival remaining at (VSGroup Treatment (%) day (D) termination Group1) 1 Vehicle — 12 None — 2NEI-01, 140 U/kg 20.8 14.5 None 0.28 3 NEI-01, 280 U/kg 37.5 16.5 None0.12

Example 11: Anticancer Activity of NEI-01 in Jurkat-Derived LeukemiaCancer Xenograft Model

The T-cell immunophenotype of acute lymphoblastic leukemia (T-ALL)accounts for about 15 to 25% of acute leukemia in adults and children.Benefitting from rapid technological advances and an emergingunderstanding, significant progress has been achieved in the treatmentof T-ALL. However, a significant number of patients remain at a highrisk for relapse, and few patients survive when the disease recurs.Thus, new therapeutic approaches are urgently needed.

Drug-induced amino acid deprivation is one strategy that has beensuccessfully used in the treatment of acute lymphoblastic leukemia,where asparaginase is an important part of induction chemotherapy.Arginine, as a precursor for initiation of a variety of metabolicpathways, has been confirmed to have a modulatory effect ontumourigenesis. Arginine deprivation has been demonstrated as apromising therapeutic approach against arginine-auxotrophic tumourswhich lack argininosuccinate synthase (ASS1), a limiting enzyme tosynthesize arginine from citrulline. This study aimed to evaluate theanticancer activity of the arginine-depriving enzyme, NEI-01 in a T-ALLJurkat xenograft model.

Human ASS1-deficient T-ALL Jurkat cells were subcutaneously inoculatedinto immunodeficient BALB/c nude mice. When the tumour volume reachedaround 40 mm³, the mice were randomly divided into two groups: a controland NEI-01 treatment group, as outlined in Table 12. The mice wereintraperitoneally (i.p.) administered with PBS or NEI-01 (5 U per mouse,˜280 U/kg) twice a week for 4 weeks. The tumour volume was measuredtwice a week.

TABLE 12 Groups and treatment regimens for Jurkat-Derived LeukemiaCancer Xenograft Model study. No. of Group Treatment Regimen animalsDuration 1 PBS, twice a week, i.p. 3 4 weeks 2 NEI-01 5 U per mouse(~280 U/kg), 3 twice a week, i.p.

The results showed that treatment with NEI-01 (5 U per mouse, ˜280 U/kg)twice a week significantly inhibited (p≤0.05) the tumour growth whencompared with the control group on Day 28 (FIG. 13).

These data provide support for the potent anticancer activity of NEI-01in a Jurkat-derived leukemia subcutaneous xenograft model.

Example 12: Determination of NEI-01 in Mice Plasma from Repeated DoseStudy

NEI-01 was administered to ICR mice by intravenously once per week for 4weeks at dosages of 160, 280 and 560 U/kg. Blood samples were taken onDay 1 and Day 22 at pre-dose (−1), 0.25, 6, 24, 48 and 72 h post-dosefor all groups on Day 1, pre-dose (−1) for all groups on Day 8 (Week 2),pre-dose (−1) for all groups on Day 15 (Week 3), pre-dose (−1), 0.25, 6,24, 48 and 72 h post-dose on Day 22 (Week 4) and before sacrificing themice on Day 29 (Week 5). 5 animals/group/sex/time point and plasmaconcentrations were quantified (FIG. 14).

The parameters for the pharmacokinetic assessment of NEI-01 for thetreatment groups and the results obtained are presented in Table 13(Day 1) and Table 14 (Day 22). All plasma concentrations of NEI-01 inthe vehicle control group were below the limit of quantification. Thus,the vehicle control group data are not presented in the tables.

TABLE 13 Pharmacokinetic parameters and measurements for Day 1 Mice forNEI-01 treatment groups. 160 U/kg 280 U/kg 560 U/kg Male Female MaleFemale Male Female C_(max) ^(a) (ng/ml) 77054 73787 108986 95395 190556213700 T_(max) ^(b) (h) 0.25 0.25 0.25 0.25 0.25 0.25 T_(1/2) ^(c) (h)21.04 31.90 36.58 25.87 35.71 34.37 AUC_(0-72 h) ^(d) 1.98 × 10⁶ 2.08 ×10⁶ 3.30 × 10⁶ 2.70 × 10⁶ 5.82 × 10⁶ 6.04 × 10⁶ (ng · h/ml) ^(a)C_(max):maximum NEI-01 concentration ^(b)T_(max): time at which C_(max) occurs^(c)T_(1/2): half-life, time taken for C_(max) to drop in half^(d)AUC_(0-72 h): the area under the curve in a plot of drugconcentration versus time from time of drug administration (time “0” totime “72 h”)

TABLE 14 Pharmacokinetic parameters and measurements for Day 22 Mice forNEI-01 treatment groups. 160 U/kg 280 U/kg 560 U/kg Male Female MaleFemale Male Female C_(max) ^(a) (ng/ml) 46607 42417 79378 68834 177988165310 T_(max) ^(b) (h) 0.25 0.25 0.25 0.25 0.25 0.25 T_(1/2) ^(c) (h)26.55 39.51 31.82 26.82 37.38 32.20 AUC_(0-72 h) ^(d) 1.65 × 10⁶ 1.67 ×10⁶ 2.83 × 10⁶ 2.04 × 10⁶ 6.27 × 10⁶ 5.97 × 10⁶ (ng · h/ml) ^(a)C_(max):maximum NEI-01 concentration ^(b)T_(max): time at which C_(max) occurs^(c)T_(1/2): half-life, time taken for C_(max) to drop in half^(d)AUC_(0-72 h): the area under the curve in a plot of drugconcentration versus time from time of drug administration (time “0” totime “72 h”)

Following intravenous administration of NEI-01 to mice, systemicexposure to NEI-01 was observed and the mean value T_(max) was 0.25 hpost-dose in both males and females on Day 1. The T_(1/2) was between21.04 and 36.58 hours.

Systemic exposure (as measured by AUC₀₋₇₂) to NEI-01 increased with dosein a proportional manner in males and females on both Day 1 and Day 22.The AUC₀₋₇₂ was similar in males and females on Day 1 (5.82×10⁶ ng·h/ml˜6.04×10⁶ ng·h/ml) and Day 22 (5.97×10⁶ ng·h/ml ˜6.27×10⁶ ng·h/ml) at560 U/kg. Also, C. results were similar to AUC₀₋₇₂ in that results weresimilar for males and females on both Day 1 and Day 22. There was alsono significant difference in body weight between males and femaletreatment groups.

INDUSTRIAL APPLICABILITY

The objective of the presently claimed invention is to providealternative methods for treating AML.

1. A method for treating acute myeloid leukemia (AML) in a subject inneed thereof, said method comprising administering a therapeuticallyeffective amount of an arginine-depleting agent to the subject, whereinthe AML is of the French-American-British (FAB) subtype M0(undifferentiated acute myeloblastic leukemia), M2 (acute myeloblasticleukemia with maturation), M4 (acute myeloblastic leukemia withmaturation), M4 eos (acute myelomonocytic leukemia with eosinophilia),M5 (acute monocytic leukemia), M6 (acute erythroid leukemia) or M7(acute megakaryoblastic leukemia).
 2. The method according to claim 1,wherein the arginine-depleting agent comprises an arginine catabolicenzyme.
 3. The method according to claim 2, wherein the argininecatabolic enzyme is an arginine deiminase, arginase, argininedecarboxylase or arginine 2-monooxygenase.
 4. The method according toclaim 1, wherein the arginine-depleting agent is a syntheticarginine-depleting agent.
 5. The method according to claim 1, whereinthe arginine-depleting agent comprises human serum albumin, an albuminbinding domain, an Fe region of an immunoglobulin, a polyethylene glycol(PEG) group, human transferrin, XTEN, a proline-alanine-serine polymer(PAS), an elastin-like polypeptide (ELP), a homo-amino-acid polymer(HAP), artificial gelatin-like protein (GLK), a carboxy-terminal peptide(CTP), or a combination thereof.
 6. The method according to claim 1,wherein the arginine-depleting agent comprises human serum albumin, analbumin binding domain, or a combination thereof.
 7. The methodaccording to claim 1, wherein the arginine-depleting agent comprises orconsists of an amino acid sequence having at least 95%, 96%, 97%, 98% or99% sequence identity to SEQ ID NO:
 1. 8. The method according to claim1, wherein the arginine-depleting agent comprises or consists of anamino acid sequence as defined in SEQ ID NO:
 1. 9. The method accordingto claim 1, wherein the AML is of the FAB subtype M4 or M7.
 10. Themethod according to claim 9, wherein the arginine-depleting agentcomprises or consists of an amino acid sequence having at least 95%,96%, 97%, 98% or 99% sequence identity to SEQ ID NO:
 1. 11. The methodaccording to claim 9, wherein the arginine-depleting agent comprises orconsists of an amino acid sequence as defined in SEQ ID NO:
 1. 12. Themethod according to claim 1, wherein the AML is auxotrophic forarginine.
 13. The method according to claim 1, wherein thearginine-depleting agent is administered intramuscularly, intravenously,subcutaneously or orally.
 14. The method according to claim 1, whereinthe arginine-depleting agent is administered intravenously.
 15. Themethod according to claim 1, wherein the subject is human.